Bacterial Genetics Pin Lin ( 凌 斌 ), Ph.D. Departg ment of Microbiology & Immunology, NCKU ext 5632 lingpin@mail.ncku.edu.tw References: 1. Chapters 5

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<ul><li> Slide 1 </li> <li> Slide 2 </li> <li> Bacterial Genetics Pin Lin ( ), Ph.D. Departg ment of Microbiology &amp; Immunology, NCKU ext 5632 lingpin@mail.ncku.edu.tw References: 1. Chapters 5 in Medical Microbiology (Murray, P. R. et al; 5 th edition) 2. Chapter 25 in Biochemistry (Nelson, D. et al; 4 th edition) </li> <li> Slide 3 </li> <li> Outline Introduction Replication of DNA Bacterial Transcription Other Genetic Regulation (Mutation, Repair, &amp; Recombination) </li> <li> Slide 4 </li> <li> Introduction Gene: a segment of DNA (or chromosome), the fundamental unit of information in a cell Genome: the collection of total genes in an organism Chromosome: the large DNA molecule associated with proteins or other components </li> <li> Slide 5 </li> <li> Why do we study Bacterial Genetics? Bacterial genetics is the foundation of the modern Genetic Engineering &amp; Molecular Biology. The best way to conquer bacterial disease is to understand bacteria first. </li> <li> Slide 6 </li> <li> Bacterial vs Human Chromosome E Coli: 1. Single circular chromosome, one copy (haploid) 2. Extrachromosomal genetic elements: Plasmids (autonomously self- replicating) Bacteriophages (bacterial viruses) 3. Maintained by polyamines, ex. spermine &amp; spermidine Human: 1. 23 chromosomes, two copies (diploid) 2. Extrachromosomal genetic elements: - Mitochondrial DNA - Virus genome 3. Maintained by histones </li> <li> Slide 7 </li> <li> Replication of Bacterial DNA-I Features: 1.Semi-conservative 2. Multiple growing forks 3. Bidirectional 4. Proofreading (DNA polymerase) Bacterial DNA is the storehouse of information. =&gt; Essential to replicate DNA correctly =&gt; Daughter cells </li> <li> Slide 8 </li> <li> Discovery of DNA synthesis </li> <li> Slide 9 </li> <li> Replication of Bacterial DNA-II Replication of bacterial genome requires several enzymes: - Helicase, unwind DNA at the replication origin (OriC) - Primase, synthesize primers to start the process - DNA polymerase, synthesize a copy of DNA, first found by Arthur Kornberg - DNA ligase, link two DNA fragements - Topoisomerase, relieve the torsional strain during the process, found by James Wang </li> <li> Slide 10 </li> <li> Slide 11 </li> <li> Outline Introduction Replication of DNA Bacterial Transcription Other Genetic Regulation (Mutation, Repair, &amp; Recombination) </li> <li> Slide 12 </li> <li> Transcriptional Regulation in Bacteria 1.Bacteria regulate expression of a set of genes coordinately &amp; quickly in response to environmental changes. 2.Operon: the organization of a set of genes in a biochemical pathway. 3.Transcription of the gene is regulated directly by RNA polymerase and repressors or inducers. 4.The Ribosome bind to the mRNA while it is being transcribed from the DNA. </li> <li> Slide 13 </li> <li> Lactose Operon 1.E Coli can use either Glucose or other sugars (ex: lactose) as the source of carbon &amp; energy. 2.In Glu-medium, the activity of the enzymes need to metabolize Lactose is very low. 3.Switching to the Lac-medium, the Lac-metabolizing enzymes become increased for this change. 4.These enzymes encoded by Lac operon: Z gene =&gt; b-galactosidase =&gt; split disaccharide Lac into monosaccharide Glu &amp; Gal Y gene =&gt; lactose permease =&gt; pumping Lac into the cell A gene =&gt; Acetylase </li> <li> Slide 14 </li> <li> Lactose Operon-Negative Control Negative ctrl - Repressor - Inducer (Allolactose) - Operator Lac Operon: - Lac metabolism - Under pos &amp; neg control In presence of Lactose </li> <li> Slide 15 </li> <li> Positive control Activator: CAP-cAMP (catabolite gene-activator protein) CAP RNA pol Lactose Operon-Positive Control In absence of Lactose </li> <li> Slide 16 </li> <li> Slide 17 </li> <li> Tryptophan Operon Negative control - Repressor - Corepressor (Tryptophan) - Operator </li> <li> Slide 18 </li> <li> Attenuation Transcription termination signal Couple Translation w/ Transcription Sequence 3:4 pair -G-C rich stem loop - Called attenuator -Like transcriptional terminator Sequence2: 3 pair - weak loop wont block translation </li> <li> Slide 19 </li> <li> Outline Introduction Replication of DNA Bacterial Transcription Other Genetic Regulation (Mutation, Repair, &amp; Recombination) </li> <li> Slide 20 </li> <li> Types of mutations 1. Base substitutions Silent mutation No change of amino acid Missense mutation Switch to another amino acid Nonsense mutation Change to a stop codon 2. Deletion &amp; Insertion - Change more base pairs in DNA =&gt; frameshift =&gt; truncated gene product 3. Rearrangements - Duplication, Inversion, Transposition </li> <li> Slide 21 </li> <li> Induced mutations Physical mutagens: e.g., UV irradiation (heat, ionizing radiation) Chemical mutagens Base analog Frameshift intercalating agents Base modification Transposable elements </li> <li> Slide 22 </li> <li> DNA Repair 1. Direct DNA repair (e.g., photoreactivation) 2. Excision repair Base excision repair Nucleotide excision repair 3. Post-replication or Recombinational repair 4. SOS response: induce many genes 5. Error-prone repair: fill in gaps with random sequences Thymine-thymine dimer formed by UV radiation </li> <li> Slide 23 </li> <li> Excision repair Nucleotide excision repair Base excision repair </li> <li> Slide 24 </li> <li> Double-strand break repair (postreplication repair) </li> <li> Slide 25 </li> <li> 1.Inducible system used only when error-free mechanisms of repair cannot cope with damage 2.Insert random nucleotides in place of the damaged ones 3.Error-prone SOS repair in bacteria </li> <li> Slide 26 </li> <li> Mechanisms of gene transfer Transformation: uptake of naked exogenous DNA by living cells. Conjugation: mediated by self-transmissible plasmids. Transduction: phage-mediated genetic recombination. Transposons: DNA sequences that move within the same or between two DNA molecules </li> <li> Slide 27 </li> <li> Importance of gene transfer to bacteria Gene transfer =&gt; a source of genetic variation =&gt; alters the genotype of bacteria. The new genetic information acquired allows the bacteria to adapt to changing environmental conditions through natural selection. Drug resistance (R plasmids) Pathogenicity (bacterial virulence) Transposons greatly expand the opportunity for gene movement. </li> <li> Slide 28 </li> <li> Slide 29 </li> <li> Natural transformation Transformation Artificial transformation (conventional method and electroporation) </li> <li> Slide 30 </li> <li> Demonstration of transformation Avery, MacLeod, and McCarty (1944) </li> <li> Slide 31 </li> <li> Gene exchange by Plasmids Plasmid Extrachromosomal Autonomously replicating Circular or linear (rarely) May encode drug resistance or toxins Various copy numbers Some are self-transmissible </li> <li> Slide 32 </li> <li> Conjugation mediated by self-transmissible plasmids (e.g., F plasmid; R plasmids) </li> <li> Slide 33 </li> <li> F plasmid Hfr strain F plasmid F plasmid can integrate into bacterial chromosome to generate Hfr (high frequency of recombination) donors Excision of F plasmid can produce a recombinant F plasmid (F) which contains a fragment of bacterial chromosomal DNA F plasmid --an episome </li> <li> Slide 34 </li> <li> Transduction phage-mediated genetic recombination Generalized v.s. specialized transduction </li> <li> Slide 35 </li> <li> Transposons Mobile genetic elements May carry drug resistance genes Sometimes insert into genes and inactivate them (insertional mutation) </li> <li> Slide 36 </li> <li> Trans-Gram gene transfer Spread of transposon throughout a bacterial population </li> <li> Slide 37 </li> <li> Mechanisms of evolution of Vancomycin- resistant Staphylococcus Aureus </li> <li> Slide 38 </li> <li> Cloning Cloning vectors plasmids phages Restriction enzymes Ligase In vitro phage packaging </li> <li> Slide 39 </li> <li> Slide 40 </li> <li> Library construction Genomic library cDNA library </li> <li> Slide 41 </li> <li> 1. Construction of industrially important bacteria 2. Genetic engineering of plants and animals 3. Production of useful proteins (e.g. insulin, interferon, etc.) in bacteria, yeasts, insect and mammalian cells 4. Recombinant vaccines (e.g. HBsAg) Applications of genetic engineering </li> <li> Slide 42 </li> <li> The End &amp; Thank You Take-Home Question: Mutations are good or bad for bacteria </li> <li> Slide 43 </li> <li> Mechanism of Recombination Homologous recombination Site-specific recombination Transposition Illegitimate recombination Intermolecular Intramolecular Double crossover Homologous recombination </li> <li> Slide 44 </li> <li> E Conjugational transposon </li> <li> Slide 45 </li> <li> Trans-Gram gene transfer Spread of transposon throughout a bacterial population </li> <li> Slide 46 </li> <li> Cloning Cloning vectors plasmids phages Restriction enzymes Ligase In vitro phage packaging </li> <li> Slide 47 </li> <li> Library construction Genomic library cDNA library </li> <li> Slide 48 </li> <li> Applications of genetic engineering Construction of industrially important bacteria Genetic engineering of plants and animals Production of useful proteins (e.g. insulin, interferon, etc.) in bacteria, yeasts, insect and mammalian cells Recombinant vaccines (e.g. HBsAg) </li> <li> Slide 49 </li> <li> Bacteriophage (bacterial virus) Icosahedral tailess Icosahedral tailed Filamentous Structure and genetic materials of phages Coat (Capsid) Nucleic acid </li> <li> Slide 50 </li> <li> Lysogenic phaseLytic phase Life cycle Phage as an example </li> <li> Slide 51 </li> <li> Virulent phages: undergo only lytic cycle Temperate phages: undergo both lytic and lysogenic cycles Plaques: a hollow formed on a bacterial lawn resulting from infection of the bacterial cells by phages. </li> </ul>

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